The overall goal of this proposed research is to investigate specific metal binding sites in large RNA molecules and their influence on RNA function. RNA displays a rich array of cellular functions involving complex interactions with other nucleic acids and proteins. RNA-metal interactions are critical to both structure and function, and understanding this influence requires effective, direct probes for metal site population and metal-induced activity and conformational changes. In work carried out under this project, the influence of both Mg2+ and Mn2+ on metal-binding and RNA structure will be investigated. Direct measurements of the metal coordination environment in RNA will be performed using Mn2+ as an EPR- detectable spectroscopic probe. Insight into RNA conformational changes will be sought using site-specific, EPR-active nitroxide spin labels. Taken together, these studies will provide important information about RNA structure as a function of solution ionic conditions, and will provide new tools for the investigation of more elaborate and conformationally dynamic RNA complexes. Specific aims of the current proposal include: 1. Investigating the unique 'metal ion core'from the Group I Intron, with a goal of understanding the specific contributions of metal sites to folding and stability in this structure 2. Determining the electrostatic requirements for folding of a 3-helix junction in the extended Hammerhead ribozyme, which will increase understanding of fundamental properties of RNA folding and also aid in separating cation requirements for folding and for catalysis in this ribozyme. 3. Determining the roles of metal ions in the Hammerhead ribozyme cleavage reaction, using targeted spectroscopic methods to define precisely metal-RNA interactions. Ribonucleic acid (RNA) is arguably the most important molecule in the cell. RNA is involved in regulating genes at all levels, and new roles for RNA are still being discovered. RNA-based chemical reactions are at the center of the most important cellular machines, the ribosome (protein synthesis) and spliceosome (gene splicing). It is therefore critical to understand the basic properties of RNA and how they are influenced by the environment.